A Colorimetric Enzyme-Linked Immunosorbent Assay with CuO ... - MDPI

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Dec 20, 2018 - on the Growth of Gold Nanoparticles In Situ. Dehua Deng 1,3, Yuanqiang Hao 2, Jiajia Xue 3, Xiuhua Liu 1,*, Xinyue Xu 3 and Lin Liu 2,3,*. 1.
nanomaterials Article

A Colorimetric Enzyme-Linked Immunosorbent Assay with CuO Nanoparticles as Signal Labels Based on the Growth of Gold Nanoparticles In Situ Dehua Deng 1,2 , Yuanqiang Hao 3 , Jiajia Xue 2 , Xiuhua Liu 1, *, Xinyue Xu 2 and Lin Liu 2,3, * 1 2 3

*

College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475001, Henan, China; [email protected] Henan Province of Key Laboratory of New Optoelectronic Functional Materials, Anyang Normal University, Anyang 455000, Henan, China; [email protected] (J.X.); [email protected] (X.X.) Henan Key Laboratory of Biomolecular Recognition and Sensing, College of Chemistry and Chemical Engineering, Shangqiu Normal University, Shangqiu 476000, Henan, China; [email protected] Correspondence: [email protected] (X.L.); [email protected] (L.L.); Tel.: +86-372-330-0925 (L.L.)

Received: 20 November 2018; Accepted: 15 December 2018; Published: 20 December 2018

 

Abstract: A colorimetric immunoassay has been reported for prostate-specific antigen (PSA) detection with CuO nanoparticles (CuO NPs) as signal labels. The method is based on Cu2+ -catalyzed oxidation of ascorbic acid (AA) by O2 to depress the formation of colored gold nanoparticles (AuNPs). Specifically, HAuCl4 can be reduced by AA to produce AuNPs in situ. In the presence of target, CuO NPs-labeled antibodies were captured via the sandwich-type immunoreaction. After dissolving CuO nanoparticles with acid, the released Cu2+ catalyzed the oxidation of AA by O2 , thus depressing the generation of AuNPs. To demonstrate the accuracy of the colorimetric assay, the released Cu2+ was further determined by a fluorescence probe. The colorimetric immunoassay shows a linear relationship for PSA detection in the range of 0.1~10 ng/mL. The detection limit of 0.05 ng/mL is comparable to that obtained by other CuO NPs-based methods. The high throughput, simplicity, and sensitivity of the proposed colorimetric immunoassay exhibited good applicability for assays of serum samples. Keywords: colorimetric immunoassay; CuO nanoparticles; gold nanoparticles; ascorbic acid; fluorescence immunoassay

1. Introduction Biosensors have been developed for detection of various analytes in the fields of clinical diagnostics, food industry, pharmaceutical chemistry, and environmental science. As to the recognition elements, antibodies are the most commonly used biorecognition molecules in construction of biosensors although many efforts have being made to replace antibodies with alternative recognition molecules [1–3]. Thus, immunoassays are still the most widespread analytical methods for the selective and sensitive detection of targets. For example, enzyme-linked immunosorbent assay (ELISA) represents the most popular technique of immunoassays in many fields. However, there still remain some disadvantages about classical ELISA assays, including the complicated and time-consuming implementation procedure, the use of enzyme-labeled, fluorescent or chemiluminescent antibodies, and the bulky measurement instrument. Therefore, numerous attempts have being made to improve the conventional immunosensing concepts [4]. To replace the classic enzyme labels, nanomaterials as signal reporters have attracted tremendous attention in the development of immunosensors, which include metal or metallic oxides, metal-organic frameworks (MOFs), luminescent nanocrystals, etc. [5–8] After being specifically captured on the Nanomaterials 2019, 9, 4; doi:10.3390/nano9010004

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sensing interface, the nanolabels can produce a detectable signal directly or be converted into the respective metal ions that can be determined by electric or optical techniques. Since each nanolabel contains large numbers of detectable atoms, the latter is more promising for the construction of highly sensitive immunosensors [9]. For example, CuO nanoparticles have been recently employed for the signal probes of immunosensors because of their advantages of low cost and good stability. After dissolving CuO nanoparticles with acid, the released Cu2+ ions can be determined by electric or optical techniques [10–16]. Among them, the fluorescence assays show high sensitivity. The released Cu2+ ions can be directly quantified with fluorescent dyes, quantum dots and nanomaterials or be indirectly determined based on the copper-catalyzed generation of fluorescent molecules [10–14]. In contrast to fluorescence assays, colorimetric assays exhibit high simplicity and require minimum instrumental investment despite their comparatively low sensitivity [17–19]. For example, based on the Cu+ -catalyzed click chemistry, Qu et al. reported a colorimetric immunoassay using azide- and alkyne-modified gold nanoparticles (AuNPs) as the probes [18]. In view of the peroxidase-like catalytic activity of Cu2+ to catalyze H2 O2 -mediated oxidation of 3, 3’, 5, 5’-tetramethylbenzidine (TMB), Zheng et al. developed an immunosensor by monitoring the generation of colored oxidation product of TMB [19]. The signal has been amplified by the Cu2+ /Cu+ -catalyzed reaction. However, in the AuNPs-based immunoassay, the AuNPs need to be prepared and modified with double recognition elements. For the Cu2+ -catalyzed oxidation of TMB system, high concentration of Cu2+ is required to produce colored products. Therefore, there still remains room to develop simple and sensitive colorimetric immunosensors with CuO NPs labels. Free Cu2+ ions can catalyze the oxidation of ascorbic acid (AA) by O2 ; AA as a reducing regent can reduce HAuCl4 into AuNPs [20,21]. Based on these facts, we have developed a protease biosensor in that peptide with an amino terminal copper and nickel-binding (ATCUN) motif can inhibit the Cu2+ -catalytic reaction by complexation with Cu2+ to allow for the AA-regulated growth of AuNPs in situ [22]. In view of the high extinction coefficient of AuNPs, herein, we developed an immunosensor by monitoring the generation of AuNPs, which is mediated by the Cu2+ -catalytic oxidation of AA. Moreover, ATCUN peptide binds to Cu2+ with high affinity (10−16 M), and Cu2+ can cause the fluorescent quenching of fluorophore by electron or energy transfer when binding to the recognition unit [23–25]. The released Cu2+ ions from the CuO NPs labels were further quantified by a fluorescently-labeled ATCUN peptide probe. The analytical performances of the colorimetric and fluorescent methods were compared with those achieved by other CuO NPs-based immunosensors. 2. Materials and Methods 2.1. Chemicals and Materials Prostate-specific antigen (PSA) antigen, first and second PSA antibodies (Ab1 and Ab2) and ELISA kits were ordered from Linc-Bio Science Co. LTD (Shanghai, China). CuO NPs were obtained from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). Bovine serum albumin (BSA), immunoglobin G (IgG), albumin and hemoglobin were obtained from Sigma-Aldrich (Shanghai, China). Fluorescently-labeled peptide SGHK-Dns was ordered from ChinaPeptides Co., Ltd. (Shanghai, China). Serum sample from one 36-year-old donor was provided by the health center of Anyang Normal University (Anyang, China). All the other chemicals were purchased from Aladdin Reagent Company (Shanghai, China). They are of analytical grade and used as received without additional purification. The solutions were prepared freshly with deionized water treated using a Millipore Milli-Qwater. 2.2. Labeling of Antibody with CuO NPs The procedure for labeling of antibody with CuO NPs follows that of the previously reported methods with slight modification [14,18]. Briefly, 0.5 mg of CuO NPs were dispersed in 0.5 mL of 10 mM phosphate buffer saline (PBS, pH 7.4) by 10-min sonication. Then, 10 µL of the Ab2 (1 mg/mL) was added to the CuO NPs solution. The suspension was shaken slightly for 120 min, followed by

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centrifugation at 5000 rpm for 5 min. The precipitate was then washed three times with PBS to remove unlabeled Ab2. The resulting CuO NPs-antibody conjugates (Ab2-CuO NPs) were dispersed with 0.2 mL of PBS containing 0.1% BSA and shaken slightly for 30 min. The mixture was then centrifuged and washed to remove free BSA. Finally, the obtained Ab2 -CuO NPs were re-dispersed in 1 mL of PBS and stored at 4 ◦ C for use. 2.3. Procedure for PSA Detection 40 µL of PBS or serum sample containing PSA was added to the ELISA plate and incubated at 37 ◦ C for 1 h. After washing the plate with the diluted cleaning solution five times, 40 µL of the prepared Ab2-CuO NPs suspension was added and incubated for 1 h again. This step was followed by washing the plate with deionized water five times to remove unbound Ab2-CuO NPs. Next, 100 µL of 10 mM HCl was added to the plate to shake for 5 min. To prevent the generated AuNPs from adhering on the plate, the colorimetric assay was conducted on a centrifuge tube. Briefly, 100 µL of PBS (20 mM, pH 7.2) was first added to the above plate to bring the final pH to about 7.0. Then, 50 µL of 0.75 mM AA stock solution was added to the plate for 30-min incubation. Next, 200 µL of the solution was taken out and mixed with 25 µL of 5 mM hexadecyltrimethylammonium chloride (CTAC) solution, and then 25 µL of 2 mM HAuCl4 was added to the mixed solution in batches. The color change was observed by the naked eye and the absorption spectra were collected on a Cary 60 UV-Vis spectrophotometer. For the fluorescent assay of the released Cu2+ , 100 µL of PBS containing 4 µM probe was added to the plate following the addition of HCl. Then, the solution was taken out and measured on the FLS980 Steady State Fluorescence and Phosphorescence Lifetime Spectrometer with an excitation wavelength of 340 nm. 2.4. Assay of PSA with ELISA Kits 40 µL of standard PSA sample or serum sample was added to the ELISA plate and incubated at 37 ◦ C for 1 h. After washing the plate with the diluted cleaning solution five times, 100 µL of the HRP-labeled antibody (HRP-Ab) solution was added and incubated for 1 h again. Then, the plate was washed with the diluted cleaning solution five times to remove unbound HRP-Ab. Next, 100 µL of the mixed solution of TMB and H2 O2 was added to the plate to incubated for 15 min, which is followed by the addition of 50 µL of stopping solution. Finally, the signal intensity at 450 nm was recorded with an ELISA microplate reader. 3. Results and Discussion 3.1. Detection Principle The detection principle of our immunosensor with antibodies-modified CuO NPs as labels is presented in Figure 1, which follows the classical sandwich structure. PSA, a biomarker of prostate diseases such as prostate cancer, prostatitis and benign prostatic hyperplasia, is tested as the model analyte. The immunoassay was carried out on a solid/liquid interface of 96-well plate. The secondary anti-PSA was labeled with CuO NPs (Ab2-CuO NPs). In the detection step, the captured CuO NPs are first dissolved by HCl to produce a lot of Cu2+ ions (Figure 1a). AA can be directly oxidized into dehydroascorbic acid (DA) by Cu2+ ; then, the resulting Cu+ is rapidly oxidized into Cu2+ by O2 . This Cu2+ -initiated redox cycling promotes the consumption of AA and thus depresses the AA-regulated generation of AuNPs. Because the color change of the generated AuNPs can be readily observed with naked eyes, no instrument is required for the readout. The amount of the released Cu2+ or the captured CuO NPs is dependent upon the target concentration. Thus, the solution color and absorbance signal change induced by the Cu2+ -catalyzed reaction can be used for the quantitative immunoassays. To demonstrate the accuracy and sensitivity of the colorimetric assay, the released Cu2+ was simultaneously quantified with a fluorescence probe. Because Cu2+ exhibits both high binding affinity to ATCUN peptide and high quenching ability towards the Dns group, an ATCUN peptide of

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SGHK labeled with a fluorophore Dns on the side chain of lysine (K) residue was used as the Cu2+ 2+ -peptide complex, sensing probe (Figure 1b).asAfter formation of the1b). CuAfter residue was used(denoted as the Cuas2+ SGHK-Dns) sensing probe (denoted SGHK-Dns) (Figure formation of 2+-peptide complex, the fluorescence of SGHK-Dns would be quenched. the Cu the fluorescence of SGHK-Dns would be quenched.

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Figure Schematic representation the immunosensor with CuO nanoparticle (CuO NP) labels Figure 1. 1. (a)(a) Schematic representation ofof the immunosensor with CuO nanoparticle (CuO NP) labels 2+ basedon on the the Cu (AA) oxidation and inand situ in growth AuNPs;of(b)AuNPs; Fluorescence based Cu2+-catalyzed -catalyzedascorbic ascorbicacid acid (AA) oxidation situ of growth (b) 2+ with the probe of SGHK-Dns. detection of the released Cu 2+ Fluorescence detection of the released Cu with the probe of SGHK-Dns.

3.2. Optimization of Experimental Conditions 3.2. Optimization of Experimental Conditions AA concentration and solution pH play decisive roles in the generation of AuNPs. Our early AA concentration and solution pH play decisive roles in the generation of AuNPs. Our early investigations have demonstrated that HAuCl4 can be reduced to AuNPs by AA at neutral pH and investigations have demonstrated that HAuCl4 can be reduced to AuNPs by AA at neutral pH and Cu2+ at micromolar concentration can catalyze the exhaustion of 200 µM AA within 10 min [22]. In the 2+ Cu at micromolar concentration can catalyze the exhaustion of 200 μM AA within 10 min [22]. In present work, the optimized experimental conditions for AuNPs generation followed those of our early the present work, the optimized experimental conditions for AuNPs generation followed those of work. To demonstrate the binding stoichiometry and fluorescence quenching efficiency of Cu2+ to the our early work. To demonstrate the binding stoichiometry and fluorescence quenching efficiency of2+ peptide probe, the fluorescence spectra of SGHK-Dns in the presence of various concentrations of Cu Cu2+ to the peptide probe, the fluorescence spectra of SGHK-Dns in the presence of various were collected. As shown in Figure 2a, the fluorescence signal of the peptide decreased gradually with concentrations of Cu2+2+ were collected. As shown in Figure 2a, the fluorescence signal of the peptide the increase of Cu concentration. The value reached to the minimum in the presence of 1 equiv of decreased gradually with the increase of Cu2+ concentration. The value reached to the minimum in Cu2+ , which is indicative of a 1:1 binding ratio (Figure 2b). The result also demonstrated that labeling the presence of 1 equiv of Cu2+, which is indicative of a 1:1 binding ratio (Figure 2b). The result also of Dns group on the side chain of lysine residue did not decrease the binding affinity of ATCUN demonstrated that2+labeling of Dns group on the side chain of lysine residue did not decrease the peptide with Cu . Moreover, the quenching efficiency was calculated to be 84.8% with the formula binding affinity of ATCUN peptide with Cu2+. Moreover, the quenching efficiency was calculated to (1 − F’/F0 ) × 100%, where F’ and F0 represent the fluorescence intensity of the probe at 552 nm with be 84.8% with the formula (12+− F’/F0) × 100%, where F’ and F0 represent the fluorescence intensity of and without addition of Cu , respectively. The high quenching efficiency can be attributed the strong the probe at 552 nm with and without addition of Cu2+, respectively. The high quenching efficiency interaction and high quenching ability of Cu2+ to the fluorescently-labeled peptide. Thus, the designed can be attributed the strong interaction and high 2+quenching ability of Cu2+ to the peptide probe can be used for the sensitive detection of Cu . fluorescently-labeled peptide. Thus, the designed peptide probe can be used for the sensitive detection of Cu2+.

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[Cu ] / μM λ / nm Figure 2. The emission spectra (a) and fluorescence intensity (b) at 552 nm of 2 μM SGHK-Dns in the 2+ (from topintensity Figure The emission spectra fluorescence (b) 552 nm of SGHK-Dns in the to bottom: 0.01, 2, 2.5 and 5 μM). presence different concentrations of Cu Figure 2. 2.of The emission spectra (a) (a) and and fluorescence intensity (b)0,at at 5520.1, nm0.5, of 221,μM µM SGHK-Dns in the 2+2+ (from top to bottom: 0, 0.01, 0.1, 0.5, 1, 2, 2.5 and 5 μM). presence of different concentrations of Cu presence of different concentrations of Cu (from top to bottom: 0, 0.01, 0.1, 0.5, 1, 2, 2.5 and 5 µM). 3.3. Feasibility 3.3. Feasibility The feasibility of our strategy was investigated by monitoring the change in color and ofof ourour strategy was investigated by monitoring the and absorption The feasibility feasibility strategy was investigated by monitoring the inchange in3a, color and absorption spectra of the detection solutions at various conditions. Aschange shown incolor Figure without spectra of the detection solutions at various conditions. As shown in Figure 3a, without Ab2-CuO NPs absorption spectra of the detection solutions at various conditions. As shown in Figure 3a, without Ab2-CuO NPs (curve/tube 1) or PSA (curve/tube 2) incubation step, the solution kept red color and (curve/tube 1) or PSA (curve/tube 2) (curve/tube incubation step, the solution kept red color and Ab2-CuO NPs (curve/tube 1) ornm PSA 2) incubation step, the solution keptthe redabsorbance color and the absorbance intensity at 530 (A 530) is high. The absorption peak can be ascribed to the surface intensity at 530 nm (A ) is high. The absorption peak can be ascribed to the surface plasmon the absorbance intensity at 530 nm (A 530 ) is high. The absorption peak can be ascribed to the surface 530 plasmon resonance of the generated AuNPs. This result indicated that AA was not oxidized and resonance of the generated AuNPs. This result indicated that AA wasthat not AA oxidized andPSA more AuNPs plasmon resonance of the generated AuNPs. This resultNPs indicated was not oxidized and more AuNPs were generated in the absence of Ab2-CuO or PSA. However, in the detection were generated in generated the absence of Ab2-CuO NPs or PSA. However, in the PSA detection system, more AuNPs were in the absence Ab2-CuO NPs or PSA. in the PSAindicating detection system, the solution was colourless and theof absorbance intensity wasHowever, greatly decreased, the solution wasAuNPs colourless andgenerated the absorbance intensity greatly decreased, indicating that no of or system, solution waswere colourless and the absorbance intensity was greatly decreased, indicating that no the or less (curve/tube 3).was This result indicated that the change less AuNPs were generated (curve/tube 3). This result indicated that the change of solution color and that no orcolor less and AuNPs were generated (curve/tube 3). This result indicated of solution absorbance intensity is dependent upon PSA capture that andthe thechange specific absorbance intensity is dependent upon PSA capture and the specific antibody-antigen interaction. solution color and absorbance intensity is dependent upon PSA capture and the specific antibody-antigen interaction. To further prove that the signal change of the colorimetric assay is 2+ ions To further that theCu signal the colorimetric assaychange is caused the released Cu 2+ ions antibody-antigen interaction. Tochange further prove that thefluorescence signal of by the colorimetric assay is caused by prove the released from of Ab2-CuO NPs, assays with SGHK-Dns as the 2+ detection probe were carried out. 2+ from Ab2-CuO NPs, fluorescence assays with SGHK-Dns as the Cu 2+ detection caused by the probe released Cu carried ions from Ab2-CuO fluorescence assays with SGHK-Dns the Cu were out. As shown NPs, in Figure 3b, the fluorescence signal in theasPSA 2+ As shown in Figure 3b, the fluorescence signal in the PSA detection system is significantly lower than Cu detection probe were carried out. As shown in Figure 3b, the fluorescence signal in the PSA detection system is significantly lower than that in the case without PSA or Ab2-CuO NPs capture. that result in thesystem case without PSA Ab2-CuO NPs capture. is consistent with thatNPs achieved by detection is significantly lower than that in colorimetric the The caseresult without PSA or Ab2-CuO capture. The is consistent withor that achieved by the assay. Thus, the proposed method 2+ the colorimetric assay. Thus, the target-induced 2+ proposed The result is target-induced consistent withCu that achievedmethod by change the based colorimetric assay. Thus, theCu proposed method based on the concentration canon bethe used for development ofconcentration colorimetric 2+ concentration change can be used for development of colorimetric or fluorescence immunosensors. based on the target-induced Cu change can be used for development of colorimetric or fluorescence immunosensors. or fluorescence immunosensors. 1

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Figure 3. 3. The TheUV-Vis UV-Vis absorption spectra and photographic images inset) (a) and emission absorption spectra and photographic images (the inset)(the (a) and emission spectra (b) for Figure The UV-Vis absorption spectra and photographic images (the 2inset) (a)2,(PSA); and emission spectra (b) for different detection Curve or tubeantigen 1, prostate-specific antigen curve 2 different3. detection systems. Curve or systems. tube 1, prostate-specific (PSA); curve or tube Ab2-CuO NPs, spectra (b) detection systems. Curve tube 1, prostate-specific (PSA); curveof2 or tube 2, Ab2-CuO PSA; curve 3 or The tube 3, or PSA + Ab2-CuO NPs.was The50antigen final concentration PSA; curve 3for or different tube 3,NPs, PSA + Ab2-CuO NPs. final concentration of PSA ng/mL. or tube PSA was2,50Ab2-CuO ng/mL. NPs, PSA; curve 3 or tube 3, PSA + Ab2-CuO NPs. The final concentration of PSA was 50 ng/mL.

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3.4. Sensitivity The sensitivity of the immunosensor was investigated by monitoring the color and signal change of the detection system in the presence of different concentrations of PSA. As shown in Nanomaterials 2018, 8, x FOR PEER REVIEW 6 of 10 Nanomaterials 2018, 8, xconcentration FOR PEER REVIEW resulted in the color change from red to colourless 6 of 10 and the Figure 4, increasing PSA 3.4. Sensitivity gradual decrease in the absorbance intensity at 530 nm. The A530 value decreased linearly with 3.4. Sensitivity increasing PSAThe concentration from 0.1 to 10 ng/mL. The linear A530 = 0.667 − 0.053[PSA] sensitivity of the immunosensor was investigated by equation monitoringisthe color and signal The sensitivity of the immunosensor was of investigated by monitoringofthe color and signal change of the detection system in the presence different concentrations PSA. As shown in (ng/mL). The detection limit of this method was estimated to be 0.05 ng/mL by determining the change of the detection system in the presence of different concentrations of PSA. As shown in Figure 4, increasing PSA concentration resulted in the color change from red to colourless and the smallest concentration of PSA at which the signal is clearly distinguishable from the background. Figure 4, increasing PSA concentration resulted in the color change from red to colourless and the gradual decrease in the absorbance intensity at 530 nm. The A530 value decreased linearly with gradual decrease inmethod the absorbance intensity atby 530analyzing nm. The Athree 530 value decreased linearly PSA with samples The reproducibility of this was0.1 evaluated prepared increasing PSA concentration from to 10 ng/mL. The linear equation isfreshly A530 = 0.667 − 0.053[PSA] increasing PSA concentration from 0.1 to 10 ng/mL. The linear equation is A 530 = 0.667 − 0.053[PSA] at the same concentration. standard deviations shown as the error (ng/mL). The detection The limit relative of this method was estimated to be (RSDs, 0.05 ng/mL by determining the bars in (ng/mL). The detection limit of this method was estimated to be 0.05 ng/mL by determining the concentration of PSA atprepared which the samples signal is clearly distinguishable from the background.acceptable Figure 4b)smallest for assays of the parallel are all less than 10.2%, suggesting smallest concentration of PSA at which the signal is clearly distinguishable from the background. The reproducibility of this method was evaluated by analyzing three freshly prepared PSA samples reproducibility of the proposed immunosensor. To the accuracy of the colorimetric of thisThe method was evaluated by demonstrate analyzing three freshly prepared PSA atThe thereproducibility same concentration. relative standard deviations (RSDs, shown as the error bars insamples Figure 2+ atconcentration the same concentration. The relative standard deviations (RSDs, shown asthe the error bars inCu Figure assay, PSA was also determined by measurement of released 4b) for assays of the parallel prepared samples are all less than 10.2%, suggesting acceptable with the 4b) for assays of the parallel prepared samples are all less than 10.2%, suggesting acceptable fluorescence probe. Asof shown in Figure 5, the fluorescence decreases linearly with the reproducibility the proposed immunosensor. To demonstrateintensity the accuracy of the colorimetric reproducibility of the proposed immunosensor. To demonstrate the accuracy of the 2+colorimetric assay, PSA concentration was also determined by measurement of the released Cu with the increase ofassay, PSAPSA concentration in the range of 0.1 to 10 ng/mL with a calibration equation of concentration was also determined by measurement of the released Cu2+ with the fluorescence probe. As shown in Figure 5, the fluorescence intensity decreases linearly with the F = 422.5 −fluorescence 22.3[PSA]probe. (ng/mL). The detection limit of the fluorescent method was about 0.1 ng/mL. As shown in Figure 5, the fluorescence intensity decreases linearly with the increase of PSA concentration in the range of 0.1 to 10 ng/mL with a calibration equation of F = 422.5 increase of PSA concentration in the range of 0.1 to 10 ng/mL with a calibration equation of Fthe = 422.5 Interestingly, the colorimetric method exhibits a comparable sensitivity with that of fluorescent − 22.3[PSA] (ng/mL). The detection limit of the fluorescent method was about 0.1 ng/mL. − 22.3[PSA] (ng/mL). The detection limit of the fluorescent method 0.1 ng/mL. assay. Moreover, the detection limit of the proposed immunosensor is comparable tofluorescent or even lower than Interestingly, the colorimetric method exhibits a comparable sensitivity withwas that about of the Interestingly, the colorimetric method exhibits a comparable sensitivity with that of the fluorescent assay. Moreover, the detectionimmunosensors limit of the proposed(Table immunosensor is comparable to or even lower that achieved by other CuO-based 1). The high sensitivity of the colorimetric assay. Moreover, the detection limit of the proposed immunosensor is comparable to or even lower than that achieved by other CuO-based immunosensors (Table2+1). The high sensitivity of the immunoassay attributed to theCuO-based high catalytic activity of(Table Cu 1). to AA the high thancan thatbeachieved by other immunosensors The oxidation, high sensitivity of theextinction colorimetric immunoassay can be attributed to the high catalytic activity of Cu2+ to AA oxidation, 2+ to AA oxidation, colorimetric immunoassay can beamplification attributed to theof high catalytic activity of Cu coefficientthe of high AuNPs, and the signal CuO NPs. For the fluorescent immunoassay, extinction coefficient of AuNPs, and the signal amplification of CuO NPs. For the the high extinctionfrom coefficient of AuNPs, the signal amplification ability of CuOofNPs. For 2+ the high sensitivity thehigh high bindingand affinity and Cu to the the peptide fluorescent results immunoassay, the sensitivity results from the quenching high binding affinity and quenching fluorescent 2+immunoassay, the high sensitivity results from the high binding affinity and quenching ability of Cu to the peptide probe. We believe that the sensitivity may be improved by using more probe. We believe that the sensitivity may be improved by using more sensitive ELISA plate. ability of Cu2+ to the peptide probe. We believe that the sensitivity may be improved by using more sensitive ELISA plate. sensitive ELISA plate. (a) (a)

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Figure 5. The emission spectra (a) and fluorescence intensity (b) for assays of different concentrations PSA (from to bottom: 0, 0.1, 1,intensity 2, 5, 10, 20 and 50 ng/mL). Figure 5. The emissionofspectra (a)top and fluorescence (b) for assays of different concentrations concentrations of PSA (from top to bottom: 0, 0.1, 1, 2, 5, 10, 20 and 50 ng/mL). of PSA (from top to bottom: 0, 0.1, 1, 2, 5, 10, 20 and 50 ng/mL).

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Table 1. Comparison of analytical performances of various CuO NPs-based biosensors.

Nanomaterials 2019, 9, 4 Method Colorimetry Colorimetry Table Colorimetry Fluorescence Method Fluorescence Colorimetry Colorimetry Fluorescence Colorimetry Fluorescence Fluorescence

Fluorescence Fluorescence Fluorescence Fluorescence DPV Fluorescence AAS Fluorescence Colorimetry Fluorescence DPV Fluorescence

Target Probe for Cu2+ Detection Detection Limit Linear Range HIV AuNPs Not reported Not reported CEA DPHE pg/mL CuO NPs-based 0.05 ~ 100 ng/mL. 1. Comparison of analytical performances of 26 various biosensors. Glypican-3 TMB/H2O2 0.26 pg/mL 0.2 ~ 200 pg/mL HER2 Quinoxaline 9.65 pg/mL 5 ~ 25 pg/mL Probe for Cu2+derivative Target Detection Limit Linear Range Detection AFP triazole complex 0.012 ng/mL 0.025 ~ 5.0 ng/mL AFP 0.3 pg/mL 0.001 ~ 100 ng/mL HIV AuNPs Not reported Not reported CEA DPHE 26 pg/mL 0.05~100 CA125 0.061 mU/mL 0.0002ng/mL. ~ 100 U/mL CdTe QDs Glypican-3 TMB/H2 O2 0.26 pg/mL 0.2~200 pg/mL CA 153 0.29 mU/mL 0.0001 ~ 200 U/mL HER2 Quinoxaline derivative 9.65 pg/mL 5~25 pg/mL CEA 1.4 pg/mL 0.005 ~ng/mL 200 ng/mL AFP triazole complex 0.012 ng/mL 0.025~5.0 AFP ENFFs 8.3 pg/mL 0.01 ~ ng/mL 200 ng/mL AFP 0.3 pg/mL 0.001~100 CA125 0.061 mU/mL 0.0002~100 AFP CdS QDs 0.45 ng/mL, 1 ~ 80U/mL ng/mL CdTe QDs CA 153 0.29 7.5 ×0.0001~200 104 ~ 1.5 ×U/mL 107 particles/μL Exosome CuNPs 4.8mU/mL × 104 particles/μL CEA 1.4 pg/mL 0.005~200 ng/mL H1N1 influenza virus GCE 10−12 g/mL 10−11 ~ 10−5 g/mL AFP ENFFs 8.3 pg/mL 0.01~200 ng/mL IgG 0.19 ng/mL 1 ng/mL ~ 104 g/mL AFP CdS QDs 0.45 ng/mL, 1~80 7 particles/µL PSA AuNPs 0.05 ng/mL ng/mL Exosome CuNPs 4.8 × 104 particles/µL 7.5 × 104 ~1.50.1 × ~1010 H1N1 influenza GCE 10−12 g/mL 10−110.1 ~10~−510g/mL PSA virus SGHK-Dns 0.1 ng/mL ng/mL

AAS HIV, Colorimetry Fluorescence

IgG

1~104 g/mL

0.19 ng/mL

7 of 10 Ref. [18] [17] [19] [10] Ref. [11] [18] [17] [19] [13] [10] [11]

[12] [14] [16] [26] [12] [14] [15] This work [16] [26] This work [13]

[15]

human immunodeficiency virus; 0.05 CEA, antigen; DPHE, PSA AuNPs ng/mL carcinoembryonic 0.1~10 ng/mL This work PSA SGHK-Dns 0.1 ng/mL 0.1~10 ng/mL work 1,2-diphenyl-2-(2-(pyridin-2-yl)hydrazono)ethanone; HER2, human epidermal growth This factor HIV, human immunodeficiency virus; CEA, carcinoembryonic antigen; DPHE, receptor 2; AFP, alpha-fetoprotein; ENFFs, electrospun nanofibrous films; QDs, quantum dots; DPV, 1,2-diphenyl-2-(2-(pyridin-2-yl)hydrazono)ethanone; HER2, human epidermal growth factor receptor 2; differential pulse voltammetry; GCE, glass carbon films; electrode; atomic absorption spectrometry. AFP, alpha-fetoprotein; ENFFs, electrospun nanofibrous QDs,AAS, quantum dots; DPV, differential pulse voltammetry; GCE, glass carbon electrode; AAS, atomic absorption spectrometry.

3.5. Selectivity 3.5. Selectivity Since the colorimetric assay shows high sensitivity and does not require advanced instrument forSince signalthe readout, the selectivity for PSA was investigated by the colorimetric method. As colorimetric assay shows highdetection sensitivity and does not require advanced instrument shown in Figure 6, for the tested proteins including PSA, BSA, IgG, albumin and hemoglobin, only for signal readout, the selectivity for PSA detection was investigated by the colorimetric method. system resulted in the color change and significant decrease in the absorbance intensity. No AsPSA shown in Figure 6, for the tested proteins including PSA, BSA, IgG, albumin and hemoglobin, significant difference in the color change and absorbance intensity was observed for the only PSA system resulted in the color change and significant decrease in the absorbancenon-specific intensity. proteins even at the 10-fold higher concentration. The high selectivity can be attributed to the high No significant difference in the color change and absorbance intensity was observed for the non-specific specificity of antibody with target protein and the low nonspecific adsorption of Ab2-CuO proteins even at the 10-fold higher concentration. The high selectivity can be attributed to theNPs highon the ELISA microwells. specificity of antibody with target protein and the low nonspecific adsorption of Ab2-CuO NPs on the ELISA microwells.

0.8

1

2

3

4

5

2

3

4

5

Abs (530 nm)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

1

Figure 6. The photographic images and absorption intensity for assays of various proteins (1, PSA; Figure The4,photographic images and absorption intensity for assays various proteins PSA; 2, BSA; 3, 6. IgG; albumin; 5, hemoglobin). The final concentration of PSAof was 10 ng/mL and(1, that of 2, BSA; 3, IgG; 4, albumin; 5, hemoglobin). The final concentration of PSA was 10 ng/mL and that of other proteins is 100 ng/mL. other proteins is 100 ng/mL.

Nanomaterials 2018, 8, x FOR PEER REVIEW Nanomaterials 2019, 9, 4

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3.6. Evaluation of Serum Samples 3.6. Evaluation of Serum Samples The detection limit of our colorimetric method is lower than the normal value (